EP1292710B1 - Procede de depistage par une matrice - Google Patents

Procede de depistage par une matrice Download PDF

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Publication number
EP1292710B1
EP1292710B1 EP01943634A EP01943634A EP1292710B1 EP 1292710 B1 EP1292710 B1 EP 1292710B1 EP 01943634 A EP01943634 A EP 01943634A EP 01943634 A EP01943634 A EP 01943634A EP 1292710 B1 EP1292710 B1 EP 1292710B1
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repertoire
members
repertoires
series
molecules
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EP01943634A
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German (de)
English (en)
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EP1292710A2 (fr
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Lucy Jessica Holt
Ian Tomlinson
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Domantis Ltd
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Domantis Ltd
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Priority claimed from GB0015443A external-priority patent/GB0015443D0/en
Priority claimed from GB0026099A external-priority patent/GB0026099D0/en
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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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Definitions

  • the present invention relates to a method which can be used to screen two or more repertoires of molecules against one another and/or to create and screen combinatorial repertoires by combining two or more repertoires.
  • the invention relates to a method whereby two repertoires of molecules can be screened such that substantially all members of the first repertoire are tested against substantially all members of the second repertoire for functional interactions.
  • the invention relates to the creation and screening of antibody repertoires by combining a repertoire of heavy chains with a repertoire of light chains such that antibodies formed by the substantially all combinations of heavy and light chains can be screened against one or more target ligands.
  • mapping and sequencing of different genomes will eventually lead to the cloning of all the proteins expressed by these organisms.
  • two-dimensional screens need to be performed so that the binding of every protein to every other protein can be tested.
  • Two dimensional screens are also required for a number of other applications.
  • techniques such as mouse immunisation coupled with the production of monoclonal antibodies and in vitro selection methods such as phage display have been used to simultaneously generate many different antibodies against many different targets.
  • in vitro selection methods such as phage display
  • phage display In order to determine which antibodies bind to which targets these pools need to be deconvoluted, which requires a complex screening procedure.
  • Matrix Screening which can be used to study all possible interactions between all the members in two repertoires of molecules which removes the need to compartmentalise individual combinations of members of these repertoires.
  • a method for screening a first repertoire of members comprising polypeptides against a second repertoire of members comprising polypeptides to identify those members of the first repertoire which interact with members of the second repertoire comprising:
  • the invention in its broadest form, provides a method for screening two repertoires of molecules against one another.
  • Individual members of the two repertoires are spatially configured to enable the juxtaposition of substantially all combinations of members of both repertoires.
  • reference herein to "substantially all combinations” does not exclude that certain juxtapositions may not occur, either by chance or by design.
  • the invention does require that two repertoires of molecules be screened against each other simultaneously, and excludes the screening of a single repertoire with individual member(s) of a second repertoire.
  • “substantially all” refers to at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the members of a repertoire.
  • juxtaposition can be arrived at by, for example, creating a series of lines for each of the two repertoires, which intersect one another.
  • the lines can be straight, substantially parallel lines, or curves, or combinations thereof; the only restriction is that substantially all members of the first repertoire should be able to interact substantially all members of the second repertoire.
  • Examples of complementary configurations include straight parallel lines, disposed at an angle to straight parallel lines; concentric circles or polygons, used together with a star of radial lines.
  • the invention there is provided a method wherein members of the both the first repertoire and the second repertoire are arranged in a series of lines, each containing a member of the first or second repertoires such that the lines, corresponding to the first repertoire and those corresponding to the second repertoire are contacted with one another so that substantially all members of the first repertoire are juxtaposed with substantially all members of the second repertoire.
  • a member can mean one single member or at least one member.
  • it refers to one single member.
  • the invention also provides the use of groups consisting of more than one member of the repertoire in each line, channel or tube.
  • groups consist of 10 of fewer members, advantageously 5 or fewer, but at least 2.
  • the present invention is not only well suited to automated methods of screening but also to manual methods, where positional accuracy cannot be guaranteed and the number of dispensing events must be limited.
  • the lines can be arranged in a variety of formats and can be arranged on a single support, or a plurality of supports.
  • molecules can be manually drawn out in the form of lines on a single support, for example on a nitrocellulose membrane.
  • These lines can also be applied to suitable supports using robotic techniques, which allow the accuracy and density of arrays to be increased to great advantage in the present invention.
  • a multi-support system can be used, wherein arrays of lines are prepared on separate supports which are then juxtaposed in order to assess interaction between, the members of the repertoires.
  • the present invention can also be applied to higher dimensional arrays, for example, those with 3 dimensions.
  • three component interactions such as enzyme, substrate and co-factor can be screened using lines, channels or tubes that are arranged in 3 dimensions.
  • the three components could be antibody heavy chain, antibody light chain and antigen, and repertoires thereof can be screened in three dimensions.
  • the screening of repertoires in 2, 3 or higher dimensions according to the present invention is highly advantageous as it reduces the number of dispensing (or pipetting) events that would be required to perform a comprehensive combinatorial screen.
  • the screening of two repertoires, of, say, 300 members against one another using conventional techniques in the prior art would require at least 90,000 separate dispensing events and the screening of three repertoires, of, say, 300 members against one another would require at least 2.7 million dispensing events.
  • the present invention reduces the number of dispensing events to comprehensively screen the same repertoires to 600 or 900, respectively, a huge saving in terms of time and labour.
  • a multidimensional array can be created in a number of ways.
  • a third dimension is created by stacking filters or other such membranes and relying on capillary action for transferring molecules, or by forcing molecules through the stack by a means such as electrophoresis or osmosis or by piercing the stack or by the use of permeable filters to create the stack.
  • a third dimension can be created by stacking non-permeable layers which at the intersections of channels (for the first and second repertoires) have holes which (once the layers are stacked) form an additional set of channels in a third dimension along which members of a third repertoire can pass.
  • the third dimension can be created using a block of gel or similar such substance, which can be injected with members of the first, second and third repertoires along the x, y and z faces, respectively, thus creating channels in a three-dimensional space which form the array.
  • the matrix of interactions between members of the first, second (and optionally third) repertoires of molecules can be created using a network of intersecting tubes or semi-permeable tubes laid adjacent to one another.
  • the members of the first, second (and optionally third) repertoires of molecules can be replaced over time with different members from the same repertoires so that a new combination or set of interactions can be screened.
  • the present invention can be used to rapidly screen multicomponent and multi-chain interactions, it can also be applied to the simultaneous creation and screening of combinatorial libraries of molecules, for example, antibody or T cell receptor libraries.
  • combinatorial libraries of molecules for example, antibody or T cell receptor libraries.
  • the pairings themselves can be generated according to the invention and, optionally screened against one or more target antigens.
  • 1000 heavy chains could be drawn as lines in one dimension, and a 1000 light chains can be drawn as lines in another, such that substantially all the heavy chain lines intersect substantially all the light chain lines, forming at their intersection fully functional and folded antibody molecules, which can then be screened with a Juxtaposed antigen, for example coated on a further support which is brought into contact with the intersecting heavy and light chain lines.
  • substantially all combinations of 1000 heavy chains and 1000 light chains will have been screened i.e. a total of 1 million different antibodies, using only 2000 dispensing events, rather than the 1 million that would have to be used according to screening techniques in the prior art. This provides a rapid way for 'naive' screening for specific interactions.
  • a repertoire of heavy chains and a repertoires of light chains the members (or any related member) of which have never been in contact or selected against a given target antigen (or a related target antigen thereof) can be screened against the target antigen to identify a specific binding heavy and light chain pairing.
  • a method for creating a combinatorial library of polypeptides comprising two claims, each member of which library comprises one member of a first repertoire of members comprising a heavy and/or light chain polypeptide and one member of a second repertoire of members comprising a heavy and/or light chain polypeptide, which method comprises the step of arranging the first repertoire of members in a first series of continuous lines, and said second repertoire in a second series of continuous lines, such that substantially all members of the first repertoire are juxtaposed to substantially all members of the second repertoire, thereby generating at the points of juxtaposition, a plurality of combinations of polypeptides comprising two chains, thereby creating a combinatorial library of polypeptides comprising two chains.
  • the combinatorial library is an antibody or T cell receptor library and the two repertoires consist of heavy and light chains (in the case of an antibody library) or alpha and beta chains (in the case of a T cell receptor library).
  • the combinatorial library so produced is preferably screened for interactions with more than one target molecule.
  • the target molecule can be provided in the form of a group of target molecules, or a repertoire thereof, and screened in a three-dimensional array as described herein.
  • the method according to the invention can be used such that a three-chain polypeptide library is created (and optionally screened) using first, second and third repertoires of molecules in three dimensions.
  • the pattern of interactions between the first, second (and optionally third) repertoires can be used to identity positive interactions, negative interactions, specific interactions or cross-reactive interactions, or to construct a phylogenic tree inferring the similarity between members of the first repertoire (using the pattern of interactions with the second and/or, optionally third, repertoires), of the second repertoire (using the pattern of interactions with the first and/or, optionally third, repertoires) and/or of the third repertoire (using the pattern of interactions with members the first and/or second repertoires).
  • nucleic acids themselves are arranged in lines, channels or tubes according to the invention and expressed to produce their corresponding polypeptides.
  • intersecting polypeptides from each of the two repertoires will be expressed together. This can assist their association, particularly when the association of the two repertoire members depends on co-operative folding, for example, as in the case of antibodies.
  • information regarding the interactions of members of the repertoires will be spatially linked to the genetic information which encodes them. This genetic information can be determined by calculating the co-ordinates of the interaction and isolating the corresponding nucleotide sequence data from any point on its line, channel or tube or by isolating the nucleotide sequence data from the intersection itself.
  • a method whereby one or more of the first, second and, optionally, third repertoires comprise a plurality of nucleic acid molecules which are expressed to produce their corresponding polypeptides in situ in the array.
  • one repertoire can consist of variants of a free hapten and the other repertoire can consist of selected anti-hapten antibodies.
  • the screen can be used to identify those antibodies that are competed for binding to the immobilised target hapten by binding to certain free hapten variants. In this case, the lack of binding would be considered a positive result.
  • Controls for such an experiment can include a line of water alongside the free haptens and a line of non-hapten binding antibodies alongside the and-hapten antibodies.
  • a single free hapten could be used to disrupt binding of members of a repertoire of anti-hapten antibodies to members of a repertoire of different immobilised hapten variants.
  • Other third molecules might include substances that enhance binding of the repertoire members to one another, which can be used itself in the form of a repertoire according to the invention, In this way, a target molecule could be immobilised on a solid support and intersecting repertoires of binders and binder enhancers could be brought into contact with the target molecule.
  • binders and binder enhancers could be brought into contact with the target molecule.
  • a method for screening a first repertoire of molecules against a second repertoire of molecules to identify members of the first and second repertoires whose interactions with one another are dependant on the presence or absence of a third molecule or set of molecules, comprising.
  • a method for screening a first repertoire of molecules against a second repertoire of molecules to identify those members of the first repertoire which do not interact with those members of the second repertoire comprising:
  • the method of the present invention bridges the gap between the initial identification of lead targets and molecules from very large repertoires and the final identification of targets or drugs for therapeutic intervention. This problem is addressed in the prior art by use of ELISA screening of possible positive interactants. However, protocols for ELISA are not easily automated for high throughput. The highly parallel nature of the method according to the present invention will reveal comprehensive interaction profiles for members of each repertoire.
  • one million clone antibody libraries could be created and screened using only 2,000 dispensing events.
  • complex protein-protein interaction maps can be created from enriched sources of interacting pairs, or possibly using entire proteomes together with very high density matrices according to the invention.
  • the invention also incorporates the key advantages of phage display and other expression-display techniques, namely that the nucleic acids encoding the members of a polypeptide repertoire can be spatially associated with their corresponding polypeptides and can thus be selected on the basis of the functional characteristics of the individual polypeptide.
  • phage display in which this association is achieved by compartmentalising the nucleic acids and the polypeptides using bacterial cells which display the polypeptides on their surfaces, the subject invention advantageously exploits a novel arraying strategy to provide this association.
  • the present invention can be extended beyond selection of binding activities to select any polypeptide repertoire on the basis of any functional property of the polypeptides, including enzymatic activity, conformation or any other detectable characteristic.
  • a method for screening two or more repertoires of enzymes which comprise a two or more enzymic reaction to identify those members of the first repertoire and those members of the second repertoire which together create a given product from a given substrate comprises:
  • a method for screening different cellular populations against different viral populations to identify those viral populations that infect those cellular populations comprises:
  • a method for screening different cellular fractions against one another to identify those cellular fractions that contain components which interact with components in other cellular fractions comprises:
  • a method for screening a peptide repertoire against the same peptide repertoire to identify those members of the peptide repertoire that interact with other members of the peptide repertoire comprises:
  • a method for creating (and optionally screening) a yeast two hybrid library consisting of substantially all members of a first repertoire of polypeptides paired with substantially all members of a second repertoire of polypeptides comprises:
  • Various apparatus can be supplied in association with reagents or tools for performing the screens described above.
  • oire refers to a population of diverse variants, for example polypeptide variants which differ in amino acid sequence, DNA variants that differ in nucleotide composition and/or sequence or any other type of molecule which can exist in a number of different forms.
  • a repertoire includes more than 10 different variants. Large repertoires comprise the highest number of possible variants for selection and can be up to 10 13 in size.
  • Smaller repertoires are particularly useful, especially if they have been pre-selected to enrich for a particularly useful subset (for example, antibodies that bind cell surface markers, enzymes that catalyse a certain set of reactions, proteins that bind to other proteins etc) or to remove unwanted members (such as those including stop codons, incapable of correct folding or which are otherwise inactive).
  • Such smaller repertories can comprise 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 or more polypeptides.
  • smaller repertoires comprise between 10 and 10 4 polypeptides.
  • two or more repertoires of polypeptides are screened against each other.
  • at lest 50% of the members or each repertoire are screened against each other in each screen.
  • 60%, 70%, 80%, 90%, 95% or even 100% of the members of each repertoire are so screened.
  • interact refers to any detectable interaction between the molecules which comprise the various repertoires and, optionally, any additional molecules that comprise the screen.
  • one repertoire might comprise a diverse population of antibodies and the other a diverse population of antigens, the interaction being a binding interaction.
  • the interaction can be an enzymatically-catalysed reaction, in which one repertoire is composed of enzymes and the other repertoire is composed of substrates therefor.
  • Any interaction can be assayed using the present invention, including binding interactions, DNA methylation, nucleic acid degradation, nucleic acid cleavage (single or double stranded), Signalling events, catalytic reactions, phosphorylation events, glycosylation events, proteolytic cleavage, chemical reactions, cellular infection and combinations thereof.
  • the detection of such interactions is well known in the art.
  • molecule refers to any substance which can be applied to the screen.
  • molecules can include peptides, polypeptides, nucleic acid molecules, purified proteins, recombinant proteins, amino acids, cDNAs, expressed cDNAs, oligonucleotides, nucleotides, nucleotide analogues, families of related genes or the corresponding proteins thereof, enzymes, DNA binding proteins, immunoglobulin family members, antibodies, T cell receptors, haptens, small organic molecules, non-organic compounds, metal ions, carbohydrates and combinations thereof.
  • the creation of repertoires of such molecules is well know in the art.
  • Polypeptides can refer to polypeptides such as expressed cDNAs, members of the immunoglobulin superfamily, such as antibody polypeptides or T-cell receptor polypeptides.
  • antibody repertoires can comprise repertoires comprising both heavy chain (V H ) and light chain (V L ) polypeptides, which are either pre-assembled or assembled and screened according to the present invention.
  • An antibody polypeptide, as used herein, is a polypeptide which either is an antibody or is a part of an antibody, modified or unmodified.
  • antibody polypeptide includes a heavy chain, a light chain, a heavy chain-light chain dimer, a Fab fragment, a F(ab') 2 fragment, a Dab fragment, a light or heavy chain single domain, and an Fv fragment including a single chain Fv (scFv) or a di-sulphide bonded Fv (dsFv).
  • polypeptides can refer to other polypeptides, such as enzymes, antigens, drugs, molecules involved in cell signalling, such as receptor molecules, or one or more individual domains of larger polypeptides, which are capable of an interaction with a target molecule.
  • Molecules according to the invention can be provided in cellular form, that is in the form of cells producing a molecule as described above, or in non-cellular form, that is not contained within cells.
  • Cells can be, for example, bacterial cells, lower eukaryotic cells (e.g., yeasts), or higher eukaryotic cells (e.g., insect, amphibian, avian or mammalian cells).cells. The use of yeast cells is also envisaged.
  • cellular population refers to a collection of cells.
  • the cells comprising a cellular population may all be of the same species and cell type, or they may be a mixed population.
  • One embodiment of a cellular population comprises an-essentially substantially uniform population of cells, for example mammalian fibroblasts, transformed with a library encoding variants of a given gene coding sequence.
  • viral population refers to a collection of virus particles.
  • the particles comprising a viral population may all be of the same species and strain, or they may be a mixed population.
  • One embodiment of a viral population comprises population of recombinant or randomly mutagenized particles, for example retroviral particles.
  • a viral population can comprise multiple individuals carrying variations of one or more gene coding sequences.
  • “Juxtaposition” in the context of the present invention, includes but is not limited to physical contact.
  • Two or more repertoires according to the invention can be juxtaposed such that the molecules are capable of interacting with one another in such a manner that the sites of interactions between the members of the repertoires can be correlated with their position
  • the repertoires can be juxtaposed with one another and with a target molecule such that the members of the repertoires interact with one another and then together interact with a target molecule.
  • An "array” as referred to herein, is a pro-determined spatial arrangement of the members of the repertoire.
  • the array can take any physical form.
  • the array can be created by manual or automated means and preferred arraying technologies are further described below.
  • a "dispensing event” is a single event whereby a substance is transferred from one discrete location to a second discrete location.
  • a discrete location can be in the form of a well, a tube, a channel, a spot, a line, a rectangle, a sphere, a cube etc. Examples of single dispensing events include:
  • a "matrix” in the context of the present invention is a particular kind of array which can be used to study substantially all possible interactions between substantially all the members in two or more repertoires of molecules.
  • Such matrices can comprise a series of intersecting lines, channels or tubes, each containing one or more members of the repertoires.
  • a single matrix will contain many individual lines, channels or tubes and many more intersections, or nodes.
  • enhanced means that a detected interaction is increased by at least 10% in the presence of a given molecule or molecules relative to the interaction in the absence of that molecule or molecules.
  • blocked means that a detected interaction is decreased by at least 10% in the presence of a given molecule or molecules relative to the interaction in the absence of that molecule or molecules.
  • cellular fraction means a portion of a cell lysate resulting from a cell fractionation process.
  • Non-limiting examples of cell fractionation processes include, detergent extraction, salt extraction, acid precipitation, extraction of lipid soluble components, membrane isolation, extraction of water soluble or aqueous components, nucleo/cytoplasmic fractionation, and separations based on centrifugal forces (e.g:, the S-100 fraction).
  • cell fractionation processes include nucleic acid isolation, chromatographic separation of components of cell lysate or fractionated cell lysate, preparative electrophoretic fractionation, ion exchange and affinity separations (e.g., immunoprecipitation or immunoaffinity chromatography, His/Ni++ interactions, GST/glutathione interactions, etc.).
  • Matrices according to the present invention can be generated in many different ways to screen many different interactions involving many different molecules.
  • the invention is characterised by the ability to screen substantially all combinations of members of two or more repertoires. We have shown that this can be performed using a series of intersecting lines but other approaches which allow combinatorial screening of two or more repertoires using a minimum number of dispensing events are envisaged, such as the use of intersecting channels or tubes.
  • Our method relies on the juxtaposition of continuous groupings of molecules to create a web in two or three dimensions whereby members of the different repertoires come together and potentially interact with one another. This contrasts with screening protocols in the prior art, whereby discontinuous spotting or compartmentalised wells are used to segregate individual combinations of molecules.
  • continuous lines, channels or tubes intersect one another such that individual combinations of molecules exist at their points of intersection, or nodes.
  • the molecular 'web' or 'network' thereby created can be used not only to identify specific interacting pairs, but also the overall pattern of interactions between two repertoires.
  • the information so provided can be used to compare the performance of members of either of the repertoires with one another and in particular can be used to rapidly identify cross-reactivities of individual repertoire members within the matrix.
  • Repertoires of small organic molecules can be constructed by methods of combinatorial chemistry.
  • Repertoires of peptides can be synthesised on a set of pins or rods, such as described in WO84/03564 .
  • a similar method involving peptide synthesis on beads, which forms a peptide repertoire in which each bead is an individual repertoire member, is described in U.S. Patent No. 4,631,211 and a related method is described in WO92/00091 .
  • a significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide, so as to facilitate identification of the amino acid sequence of each library member.
  • a unique identifier tag such as an oligonucleotide
  • Another chemical synthesis method involves the synthesis of arrays of peptides (or peptidomimetics) on a surface in a manner that places each distinct library member (e.g., unique peptide sequence) at a discrete, predefined location in the array.
  • the identity of each library member is therefore determined by its spatial location in the array.
  • the locations in the array where binding interactions between a predetermined molecule (e.g., a receptor) and reactive library members occur is determined, thereby identifying the sequences of the reactive library members on the basis of spatial location.
  • the present invention is especially useful for the screening of protein-protein interactions, particularly antibody-antigen interactions.
  • the preparation of appropriate antibody repertoires useful in the present invention is described in WO 99/20749 , the disclosure of which is incorporated herein by reference.
  • WO 99/20749 describes how a library of immunoglobulins can be prepared and preselected using a generic ligand, and/or prepared using a single main-chain conformation.
  • Libraries as described in WO 99/20749 can be expressed in host organisms, as described therein or according to techniques well known in the art, to produce repertoires of polypeptides which are suitable for arraying and use in the present invention.
  • polypeptides can be synthesised in situ for use in the present invention, or expressed using in vitro transcription/translation.
  • molecules can be arrayed by any one of a variety of methods, manual or automated, in order to create a matrix, depending upon whether the molecules are arrayed as such or expressed by arrayed nucleotide precursors, which may or may not be present in host cells.
  • Arrays are advantageously created by robotic means, since robotic techniques allow the creation of precise and condensed matrices, which can be easily replicated so that, for example, a combinatorial antibody repertoire created according to the invention can be screened against many different target ligands.
  • Robotic platforms are well-known in the art, and machines are available from companies such as Genetix, Genetic MicroSystems and BioRobotics which are capable of arraying at high speed with great accuracy over small or large surfaces.
  • Such machines are capable of spotting purified protein, supernatant or cells onto porous or non-pomus surfaces, such that they can subsequently be fixed thereto if necessary to produce stable arrays.
  • robotic manipulation is the preferred method for creating high density arrays
  • any technique including manual techniques, which is suitable for locating molecules or cells at predefined locations on a support, can be used.
  • Arraying can be regular, such that lines are 'drawn' at a given distance from the next, irregular or random.
  • the repertoires of molecules can be screened in solution for interactions or one or more of the repertoires can be immobilised on a solid support.
  • two solutions can flow down two channels such that at their point of intersection an interaction occurs which can be detected by, for example, a colorimetric, fluorescent, or luminescent reaction.
  • one of the repertoires could be immobilised on a nitrocellulose membrane by, for example, cross-linking and then solutions corresponding to a second repertoire could be 'drawn' onto the support harbouring the immobilised members of the first repertoire.
  • Such immobilisation can be direct or indirect
  • indirect immobilisation can involve arraying a polypeptide repertoire onto a solid support coated with a generic ligand.
  • members of the repertoires are directed to their positions by means of a tagging system, such that each line, binds or is predisposed to bind a particular member of the repertoire.
  • each polypeptide in one member of a repertoire can comprise a tag, such as an epitope for a known antibody, or a member of an affinity pair (e.g., avidin/biotin, etc.).
  • the line is coated with a corresponding molecule that binds the tag (e.g., an antibody specific for the epitope tag, or the corresponding member of the binding pair). Contacting the coated line, with a solution comprising the tagged member of the repertoire will effect the arrangement of that member on the array.
  • both repertoires could be immobilised on a separate solid supports and then juxtaposed to identify interacting pairs.
  • matrices of polypeptides can be created by first arraying their nucleic acid precursors in host cells and then by expressing the nucleotide sequences to produce the corresponding polypeptides.
  • yeast cells can be used to express one or more repertoires of molecules useful in a method according to the invention.
  • Methods of introducing and expressing exogenous nucleic acids in yeast are well known in the art.
  • One preferred approach using yeast takes advantage of yeast two-hybrid techniques. In this approach, one population of yeast is transformed with a library encoding a repertoire of fusions with one member of a two-hybrid pair, and another population is transformed with a library encoding a repertoire of fusions with the corresponding second member of a two-hybrid pair.
  • the two yeast cell populations are of different mating types.
  • the two populations are arranged so as to create an array, such that yeast cells containing all members of the first repertoire intersect with yeast cells containing all members of the second repertoire, and the cells are allowed to mate. Interactions between members of the first repertoire and the second repertoire will generate a signal from an appropriate two-hybrid reporter construct.
  • insect, amphibian, avian, mammalian or other higher eukaryotic cells can be used.
  • a repertoire of molecules small organic molecules, peptides, polypeptides, etc.
  • a repertoire of modified-recombinant cell surface receptors e.g., a receptor with a variable cassette inserted in a region instrumental in ligand binding or activation
  • Subsequent detection of receptor activation or inhibition in the cells indicates which of the molecules affected the activity of which modified receptor.
  • the process permits both the identification of new modulators of the receptor or other protein, and the rapid identification of structure/function relationships.
  • the method can also be adapted to use higher eukaryotic cells for the expression of both repertoires being analysed for interaction. This would be accomplished, for example, by expressing both repertoires as cell surface molecules, or for example, by expressing one repertoire as a secreted protein and the other as a cell surface protein. Upon contact or close juxtaposition of the cells expressing the respective repertoires, productive interaction of members of each repertoire with members of the other can be detected.
  • One skilled in the art can readily generate higher eukaryotic cells expressing a given repertoire of polypeptides.
  • Methods of detecting interactions will vary with the exact nature of the array generated. For example, methods will vary depending on whether the array uses cells or not
  • detection methods include: fluorescence resonance energy transfer (FRET); fluorescence quenching; reporter expression (e.g., luciferase, GST, chloramphenicol acetyl transferase, ⁇ -galactosidase, antibiotic resistance); rescue from auxotrophy; signalling events, such as changes in second messenger levels, GDP for GTP exchange, kinase activation or phosphorylation, phosphatase activation or dephosphorylation, proteolysis or altered ion permeability; enzymatic reactions; methylation; nucleic acid cleavage; glycosylation; proteolysis; and infection (e.g., with a virus or phage).
  • FRET fluorescence resonance energy transfer
  • reporter expression e.g., luciferase, GST, chloramphenicol acetyl transferase, ⁇
  • Molecules selected according to the method of the present invention can be employed in substantially any process.
  • the molecules are polypeptides, they can be used in any process which involve binding or catalysis, including in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.
  • antibody molecules can be used in antibody based assay techniques, such as ELISA, techniques, Western blotting, immunohistochemistry, affinity chromatography and the like, according to methods known to those skilled in the art.
  • the molecules selected according to the invention are of use in diagnostic, prophylactic and therapeutic procedures.
  • enzyme, variants generated and selected by these methods can be assayed for activity, either in vitro or in vivo using techniques well known in the art, by which they are incubated with candidate substrate molecules and the conversion of substrate to product is analysed.
  • Selected cell-surface receptors or adhesion molecules might be expressed in cultured cells which are then tested for their ability to respond to biochemical stimuli or for their affinity with other cell types that express cell-surface molecules to which the undiversified adhesion molecule would be expected to bind, respectively.
  • proteins prepared according to the invention involve the administration of polypeptides selected according to the invention to a recipient mammal, such as a human.
  • a recipient mammal such as a human.
  • Substantially pure molecules of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human.
  • the selected polypeptides can be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent staining and the like ( Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY ).
  • the selected antibodies or binding proteins thereof of the present invention will typically find use in preventing, suppressing or treating inflammatory states, allergic hypersensitivity, cancer, bacterial or viral infection, and autoimmune disorders (which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis).
  • prevention involves administration of the protective composition prior to the induction of the disease.
  • suppression refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.
  • Treatment involves administration of the protective composition after disease symptoms become manifest.
  • EAE in mouse and rat serves as a model for MS in human.
  • the demyelinating disease is induced by administration of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et al., eds., Grune and Stratton, New York, pp. 179-213 ; McFarlin et al. (1973) Sience, 179: 478 : and Satoh et al. (1987) J. Immunol., 138: 179 ).
  • the selected antibodies, receptors (including, but not limited to T-cell receptors) or binding proteins thereof of the present invention can also be used in combination with other antibodies, particularly monoclonal antibodies (MAbs) reactive with other markers on human cells responsible for the diseases.
  • MAbs monoclonal antibodies
  • suitable T-cell makers can include those grouped into the so-called "Clusters of Differentation,” as named by the First International Leukocyte Differentiation Workshop ( Bernhard et al. (1984) Leukocyte Typing, Springer Verlag, NY ).
  • the present selected antibodies, receptors or binding proteins will be utilised in purified form together with pharmacologically appropriate carriers.
  • these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.
  • Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in - suspension can be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
  • Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, can also be present ( Mack (1982) Remington's Pharmaceutical Sciences, 16th Editi on).
  • the selected polypeptides of the present invention can be used as separately administered compositions or in conjunction with other agents. These can include various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the selected antibodies, receptors or binding proteins thereof of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.
  • immunotherapeutic drugs such as cylcosporine, methotrexate, adriamycin or cisplatinum
  • Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the selected antibodies, receptors or binding proteins thereof of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target
  • the route of administration pharmaceutical compositions according to the invention of pharmaceutical compositions according to the invention can be any of those commonly known to those of ordinary skill in the art.
  • the selected antibodies, receptors or binding proteins thereof of the invention can be administered to any patient in accordance with standard techniques.
  • the administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter.
  • the dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameter to be taken into account by the clinician.
  • the selected polypeptides of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of antibody activity loss (e.g. with conventional immunoglobulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted upward to compensate.
  • compositions containing the present selected polypeptides or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
  • an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected antibody, receptor (e.g. a T-cell receptor) or binding protein thereof per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used.
  • compositions containing the present selected polypeptides or cocktails thereof can also be administered in similar or slightly lower dosages.
  • a composition containing a selected polypeptide according to the present invention can be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal.
  • the selected repertoires of polypeptides described herein can be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells.
  • Blood from a mammal can be combined extracorporeally with the selected antibodies, cell-surface receptors or binding proteins thereof whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques,
  • the two filter capture system used as part of the present example is based upon that described in our co-pending UK patent application entitled "Array Screening Method", ( UK Patent Application Number: 9928787.2 ). Bacteria are grown in lines on one filter and the scFvs thereby produced are captured on a second filter that has lines of antigen bound to the nitrocellulose, which are oriented at 90° from those lines of scFv on the first filter (see Fig 1). At intersections where scFv interacts with antigen, the scFv is captured if the antigen and scFv bind. In this example, detection of bound scFv is performed with superantigen Protein L conjugated to HRP.
  • the antigens are from human expression library hEXl, prepared from foetal brain poly(A)+ RNA by oligo(dT)-priming (Büssow et al 1998). cDNAs are cloned directionally in a modified pQE-30 vector (Qiagen).
  • Antigen clones were grown overnight in liquid culture (2xTY containing 100 ⁇ g/ml Amp, and 1 % glucose) at 37°C.
  • liquid cultures were transferred to a pre-wetted PVDF filter (soak in ethanol, rinse in PBS and dip in 2xTY) by drawing along the filter against a metal ruler with a p10 filter tip (Art) not mounted on a pipette. Thus, the capillary action of the tip was used for delivery of the liquid onto the surface of the membrane.
  • Each clone was drawn onto the filter 6 mm from the previous one.
  • liquid cultures were transferred to a PVDP filter using the robotic head depicted in Fig 3 attached to a Kaybee Systems picker/gridder system. Each clone was drawn onto the filter 4.5mm mm from the previous one at a speed of 25 mm/s.
  • the antigen filters were then grown overnight on TYE agar plates containing 100 ⁇ g/ml Amp, 1 % glucose at 30 °C. The filter was then transferred to another TYE agar plate containing 100 ⁇ g/ml Amp, 1mM IPTG for 3h at 37 °C for induction of the clones.
  • Antigen filters were removed from the plate and denatured on pre-soaked blotting paper containing 0.5M NaOH, 1.5 M NaCl for 10 min, neutralised for 2 x 5 min in 1M Tris-HCl, pH7.5, 1.5M NaCl and incubated for 15 min in 2 x SSC. Filters were dried, soaked briefly in ethanol and then blocked in 4 % Marvel PBS, rinsed in PBS and dipped in 2xTY.
  • the scFvs are from a library based on a single human framework for V H (V3-23/DP-47 and J H 4b) and V K (012/02/DPK9 and J k 1), with side chain diversity (NNK or DVT encoded) incorporated at positions in the antigen binding site that make contacts to antigen in known structures and are highly diverse in the mature repertoire.
  • the fold that is used is frequently expressed in vivo, and binds to the generic ligands Protein L and A, which facilitate capture or detection of the scFvs but do not interfere with antigen binding.
  • the scFv clones have been pre-screened in scFv format for binding to Protein A and Protein L to ensure that they were functional.
  • Antibody clones were grown overnight in liquid culture (2xTY containing 100 ⁇ g/ml ampicillin and 1% glucose) at 37°C. Liquid cultures were then transferred to a pre-blocked nitrocellulose filter (4% skimmed milk powder in PBS for 1 hour at room temperature (RT), rinse in PBS and dip in 2xTY). Manual and robotic transfer of antibodies to the filter was performed as for the antigen cultures, except that the density of scFvs lines created by robotic transfer was one every 1.125 mm.
  • ScFv filters were then grown on TYE agar plates containing 100 ⁇ g/ml Amp, 1 % glucose at 37°C. After overnight growth the antigen filter was placed onto a fresh TYE agar plate 100 ⁇ g/ml Amp, 1mM IPTG, dried, and then the scFv filter was placed on top of this. The plate with the two filters was then incubated for 3h at 30°C for induction of the scFvs.
  • the top (scFv) filter was discarded and the second (antigen) filter was washed 3 x with PBS/0.05% Tween (PBST) and blocked with 4% MPBS for 30 min at RT.
  • the filters were washed 3x with PBST and then incubated with a Protein L HRP conjugate (Actigen, 1/2000) in 4% MPBS for 1 hr at RT. Filters were then washed a further three times with PBST and developed with ECL reagent. All incubations were performed in 50 ml of buffer on a gently agitating shaker.
  • each of the four anti ubiquitin scFvs bound the two known ubiquitin clones and each of the five potential ubiquitin clones.
  • scFv Ub1b1 and scFv R14 are highly cross reactive.
  • 14 antigen clones identified in a primary antigen array screen as possible binders to twelve scFv clones C2 to H11.
  • antigen M a protein of unknown function
  • antigen E (a DNA binding protein) binds specifically to scFv H11. This demonstrates the utility of the matrix screen in confirming interactions originally identified in an antigen array screen.
  • FIG. 8 A schematic for three-dimensional screening according to the invention is shown ( Figure 8).
  • members of the repertoires are arranged in planes in the x, y and z axes and the interactions occur at the various vertices of the intersecting planes.
  • an anti-BSA heavy chain is deposited on one plane
  • an anti-BSA light chain is deposited on a second plane
  • BSA is deposited in the third plane
  • An interaction at their vertex is detected only when all three are present (Figure 9).

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Claims (49)

  1. Procédé de criblage d'un premier répertoire immunologique de membres comprenant des polypeptides sur un deuxième répertoire immunologique de membres comprenant des polypeptides pour identifier les membres du premier répertoire immunologique qui interagissent avec les membres du deuxième répertoire immunologique, comprenant :
    (a) l'agencement du premier répertoire immunologique dans au moins une première série de lignes continues dans laquelle chaque ligne de ladite première série comprend un membre dudit premier répertoire immunologique et l'agencement du deuxième répertoire immunologique dans au moins une deuxième série de lignes continues dans laquelle chaque ligne de ladite deuxième série comprend un membre dudit deuxième répertoire immunologique, dans lequel les premier et deuxième répertoires immunologiques forment au moins une matrice, de telle sorte que sensiblement tous les membres du premier répertoire immunologique soient juxtaposés sensiblement à tous les membres du deuxième répertoire immunologique ; et
    (b) la détection d'une interaction entre les polypeptides des premier et deuxième répertoires immunologiques, identifiant ainsi les membres du premier répertoire immunologique qui interagissent avec les membres du deuxième répertoire immunologique.
  2. Procédé selon la revendication 1, dans lequel lesdits premier et deuxième répertoires immunologiques sont chacun présents dans une série de lignes continues non entrecroisées.
  3. Procédé selon l'une quelconque des revendications 1 et 2, dans lequel les premier et deuxième répertoires immunologiques sont agencés sur des premier et deuxième supports respectivement.
  4. Procédé selon la revendication 1 ou 2, dans lequel les membres des premier et deuxième répertoires immunologiques sont appliqués sur un seul support.
  5. Procédé selon l'une quelconque des revendications précédentes, dans lequel chaque ligne desdits premier et deuxième répertoires immunologiques s'étend le long de canaux tracés ou gravés dans un matériau solide de telle sorte que sensiblement tous les canaux contenant un membre du premier répertoire immunologique s'entrecroisent sensiblement avec tous les canaux contenant un membre du deuxième répertoire immunologique .
  6. Procédé selon l'une quelconque des revendications précédentes, dans lequel la chaîne polypeptidique est un polypeptide à chaîne lourde ou légère.
  7. Procédé selon l'une quelconque des revendications 1 à 5, dans lequel ledit polypeptide à chaîne lourde ou légère est un fragment dAb.
  8. Procédé selon la revendication 6, dans lequel ledit premier répertoire immunologique comprend VH ou VL.
  9. Procédé selon la revendication 7, dans lequel ledit deuxième répertoire immunologique comprend VH ou VL.
  10. Procédé selon l'une quelconque des revendications 1 à 9, dans lequel ledit premier répertoire immunologique comprend VH, et ledit deuxième répertoire immunologique comprend VL.
  11. Procédé selon l'une quelconque des revendications 6 à 10, dans lequel ladite étape de détection comprend la mise en contact de ladite au moins une matrice avec un épitope cible, et la détection de la liaison de l'épitope cible par des membres juxtaposés desdits premier et deuxième répertoires immunologiques sur ladite matrice, dans lequel ladite liaison de l'antigène cible constitue une indication d'une interaction des membres desdits premier et deuxième répertoires immunologiques.
  12. Procédé selon l'une quelconque des revendications 1 à 10, dans lequel ladite étape de détection comprend la mise en contact de ladite au moins une matrice avec un troisième répertoire immunologique de membres antigènes cibles agencés dans une série de lignes continues, et la détection de la liaison de l'antigène cible par des membres juxtaposés desdits premier et deuxième répertoires immunologiques au niveau de positions sur ladite matrice, dans lequel ladite liaison de l'antigène cible constitue une indication d'une interaction des membres desdits premier et deuxième répertoires immunologiques.
  13. Procédé selon la revendication 12, dans lequel une pluralité de lignes dudit troisième répertoire immunologique comprennent un antigène cible différent.
  14. Procédé selon la revendication 12, dans lequel lesdits premier et deuxième répertoires immunologiques sont chacun agencés dans une série de lignes continues non entrecroisées.
  15. Procédé de génération d'une bibliothèque combinatoire de polypeptides comprenant deux chaînes, chaque membre de la bibliothèque comprenant un membre d'un premier répertoire immunologique de membres comprenant un polypeptide à chaîne lourde et/ou légère et un membre d'un deuxième répertoire immunologique de membres comprenant un polypeptide à chaîne lourde et/ou légère, lequel procédé comprend l'étape d'agencement du premier répertoire immunologique de membres dans une première série de lignes continues, et dudit deuxième répertoire immunologique dans une deuxième série de lignes continues, de telle sorte que sensiblement tous les membres du premier répertoire immunologique soient juxtaposés sensiblement à tous les membres du deuxième répertoire immunologique, générant ainsi aux points de juxtaposition, une pluralité de combinaisons de polypeptides comprenant deux chaînes, générant ainsi une bibliothèque combinatoire de polypeptides comprenant deux chaînes.
  16. Procédé selon la revendication 15, dans lequel la bibliothèque combinatoire produite est criblée en vue de détecter les interactions avec plusieurs molécules cibles.
  17. Procédé selon la revendication 12, dans lequel une bibliothèque de polypeptides à trois chaînes est générée (et éventuellement criblée) en utilisant les premier, deuxième et troisième répertoires immunologiques de molécules en trois dimensions.
  18. Procédé selon l'une quelconque des revendications précédentes, moyennant quoi le modèle d'interactions entre les (premier, deuxième et éventuellement troisième) répertoires immunologiques est utilisé pour identifier les interactions positives, les interactions négatives, les interactions spécifiques ou les interactions de réaction croisée ou est utilisé pour construire un arbre phylétique pour déduire la similarité entre les membres du premier répertoire immunologique (en utilisant le modèle d'interactions avec le deuxième et/ou, éventuellement, le troisième répertoire(s) immunologique(s)), du deuxième répertoire immunologique (en utilisant le modèle d'interactions avec le premier et/ou, éventuellement, le troisième répertoire (s) immunologique (s)), et/ou du troisième répertoire immunologique (en utilisant le modèle d'interactions avec les membres du premier et/ou du deuxième répertoire(s) immunologique(s)).
  19. Procédé selon la revendication 18, dans lequel lesdits premier et deuxième répertoires immunologiques sont chacun agencés dans une série de lignes continues non entrecroisées.
  20. Procédé selon la revendication 15, dans lequel lesdits polypeptides sont des chaînes polypeptidiques lourdes ou légères d'anticorps.
  21. Procédé selon la revendication 15, dans lequel lesdits polypeptides sont des fragments dAb.
  22. Procédé selon la revendication 15, dans lequel ledit premier répertoire immunologique comprend VH ou VL.
  23. Procédé selon la revendication 15, dans lequel ledit deuxième répertoire immunologique comprend VH ou VL.
  24. Procédé selon la revendication 15, dans lequel ledit premier répertoire immunologique comprend VH et ledit deuxième répertoire immunologique comprend VL.
  25. Procédé de criblage de la bibliothèque combinatoire de polypeptides à deux chaînes selon la revendication 15, pour la liaison à une molécule cible, le procédé comprenant l'étape de détection d'une interaction entre les polypeptides à deux chaînes et la molécule cible.
  26. Procédé selon la revendication 25, dans lequel ladite étape de détection comprend la mise en contact de ladite bibliothèque combinatoire de polypeptides à deux chaînes avec un troisième répertoire immunologique de membres antigènes cibles agencés dans une série de lignes continues de telle sorte qu'une pluralité de membres desdits premier, deuxième, et troisième répertoires immunologiques soient juxtaposés à une pluralité d'autres membres desdits premier, deuxième, et troisième répertoires immunologiques, et la détection de la liaison de l'antigène cible par des membres juxtaposés desdits premier et deuxième répertoires immunologiques, dans lequel ladite liaison de l'antigène cible constitue une indication d'une interaction des membres desdits premier et deuxième répertoires immunologiques.
  27. Procédé selon la revendication 26, dans lequel lesdits premier, deuxième, et troisième répertoires immunologiques sont chacun agencés dans une série de lignes continues non entrecroisées.
  28. Procédé selon l'une quelconque des revendications précédentes, moyennant quoi un ou plusieurs des premier, deuxième et, s'il est présent, troisième répertoires immunologiques sont fournis par une pluralité de séquences d'acides nucléiques qui codent pour ledit polypeptide à chaîne lourde ou légère desdits premier et deuxième répertoires immunologiques ou ledit épitope cible dudit troisième répertoire immunologique et qui sont exprimés pour produire leurs polypeptides correspondants in situ dans la matrice.
  29. Procédé selon la revendication 28, dans lequel les séquences d'acides nucléiques sont fournies par des vecteurs d'expression qui codent pour des membres polypeptidiques du répertoire immunologique, et sont fonctionnellement liées aux séquences de contrôle suffisamment pour diriger la transcription des molécules d'acide nucléique.
  30. Procédé selon la revendication 29, dans lequel le vecteur d'expression est un bactériophage.
  31. Procédé selon la revendication 29, dans lequel le vecteur d'expression est un plasmide.
  32. Procédé selon la revendication 29, dans lequel le vecteur d'expression est une molécule d'acide nucléique linéaire.
  33. Procédé selon la revendication 29, dans lequel les acides nucléiques sont contenus et exprimés au sein de cellules.
  34. Procédé selon la revendication 33, dans lequel les cellules sont choisies dans le groupe consistant en des cellules bactériennes, des cellules eucaryotes inférieures et des cellules eucaryotes supérieures.
  35. Procédé selon la revendication 28, dans lequel les molécules d'acide nucléique sont immobilisées dans la forme d'acide nucléique nu ou complexé.
  36. Procédé selon l'une quelconque des revendications précédentes, dans lequel les membres d'au moins un répertoire immunologique sont déployés en utilisant un procédé robotisé.
  37. Procédé de dépistage d'un premier répertoire immunologique de molécules sur un deuxième répertoire immunologique de molécules pour identifier les membres du premier répertoire immunologique qui n'interagissent pas avec les membres du deuxième répertoire immunologique, comprenant :
    (a) l'agencement du premier répertoire immunologique dans au moins une première série de lignes continues dans laquelle chaque ligne de ladite première série comprend un membre dudit premier répertoire immunologique et l'agencement du deuxième répertoire immunologique dans au moins une deuxième série de lignes continues dans laquelle chaque ligne de ladite deuxième série comprend un membre dudit deuxième répertoire immunologique, dans lequel les premier et deuxième répertoires immunologiques forment au moins une matrice, de telle sorte que sensiblement tous les membres du premier répertoire immunologique soient juxtaposés sensiblement à tous les membres du deuxième répertoire immunologique ; et
    (b) l'identification des membres des premier et deuxième répertoires immunologiques qui n'interagissent pas les uns avec les autres.
  38. Procédé de criblage d'un premier répertoire immunologique de molécules sur un deuxième répertoire immunologique de molécules pour identifier les membres du premier et du deuxième répertoires immunologiques dont les interactions les uns avec les autres dépendent de la présence ou de l'absence d'une troisième molécule ou d'un troisième ensemble de molécules, comprenant :
    (a) l'agencement du premier répertoire immunologique dans au moins une première série de lignes continues dans laquelle chaque ligne de ladite première série comprend un membre dudit premier répertoire immunologique et l'agencement du deuxième répertoire immunologique dans au moins une deuxième série de lignes continues dans laquelle chaque ligne de ladite deuxième série comprend un membre dudit deuxième répertoire immunologique, dans lequel les premier et deuxième répertoires immunologiques forment au moins une matrice, de telle sorte que sensiblement tous les membres du premier répertoire immunologique soient juxtaposés sensiblement à tous les membres du deuxième répertoire immunologique ; et
    (b) la détection des interactions entre les membres du premier répertoire immunologique et les membres du deuxième répertoire immunologique en présence de différentes concentrations de la troisième molécule ou du troisième ensemble de molécules.
  39. Procédé selon la revendication 38, dans lequel la troisième molécule ou le troisième ensemble de molécules interagissent avec certains membres du premier répertoire immunologique, permettant ainsi aux membres du premier répertoire immunologique d'interagir avec certains membres du deuxième répertoire immunologique.
  40. Procédé selon la revendication 38, dans lequel les interactions entre les membres du premier et du deuxième répertoires immunologiques nécessitent la liaison simultanée de ces membres à la troisième molécule ou au troisième ensemble de molécules.
  41. Procédé selon la revendication 38, dans lequel les interactions entre les membres du premier et du deuxième répertoires immunologiques sont amplifiées par la présence d'une troisième molécule ou d'un troisième ensemble de molécules.
  42. Procédé selon la revendication 38, dans lequel les interactions entre les membres du premier et du deuxième répertoires immunologiques sont bloquées par la présence d'une troisième molécule ou d'un troisième ensemble de molécules.
  43. Procédé selon la revendication 1, dans lequel il est nécessaire d'avoir moins d'événements d'administration que le nombre d'interactions à tester.
  44. Procédé de criblage de deux ou plusieurs répertoires immunologiques d'enzymes qui comprennent deux ou plusieurs réactions enzymatiques pour identifier les membres du premier répertoire immunologique et les membres du deuxième répertoire immunologique qui génèrent conjointement un produit donné à partir d'un substrat donné, lequel procédé comprend :
    (a) l'agencement du premier répertoire immunologique dans au moins une série de lignes continues dans laquelle chaque ligne de ladite première série comprend un membre dudit premier répertoire immunologique et l'agencement du deuxième répertoire immunologique dans au moins une série de deuxièmes lignes continues dans laquelle chaque ligne de ladite deuxième série comprend un membre dudit deuxième répertoire immunologique, dans lequel les premier et deuxième répertoires immunologiques forment au moins une matrice, de telle sorte que sensiblement tous les membres du premier répertoire immunologique soient juxtaposés sensiblement à tous les membres du deuxième répertoire immunologique ; et
    (b) la détection de la formation de produit aux intersections des membres du premier et du deuxième répertoires immunologiques.
  45. Procédé de criblage de différentes populations cellulaires sur différentes populations virales pour identifier les populations virales qui infectent ces populations cellulaires, lequel procédé comprend:
    (a) l'agencement des populations cellulaires et des populations virales pour former au moins une matrice, dans lequel les populations virales sont agencées dans au moins une première série de lignes continues dans laquelle chaque ligne de ladite population virale comprend un membre de ladite population et l'agencement de la population cellulaire dans au moins une deuxième série de lignes continues dans laquelle chaque ligne de ladite deuxième série comprend un membre de ladite population cellulaire, dans lequel les populations virales et cellulaires forment au moins une matrice, de telle sorte que sensiblement tous les membres de la population cellulaire soient juxtaposés sensiblement à tous les membres de la population virale ; et
    (b) la détection des populations virales qui infectent ces populations cellulaires.
  46. Procédé de criblage de différentes fractions cellulaires les unes sur les autres pour identifier les fractions cellulaires qui contiennent des composants qui interagissent avec des composants dans d'autres fractions cellulaires, lequel procédé comprend :
    (a) l'agencement des fractions cellulaires dans au moins deux séries de lignes continues dans lesquelles chaque ligne desdites première et deuxième séries comprend un membre de ladite fraction cellulaire et de telle sorte que sensiblement toutes les différentes fractions cellulaires soient juxtaposés les unes aux autres.
    (b) la détection des interactions aux intersections des différentes fractions cellulaires.
  47. Procédé de criblage d'un répertoire immunologique peptidique sur le même répertoire immunologique peptidique pour identifier les membres du répertoire immunologique peptidique qui interagissent avec d'autres membres du répertoire immunologique peptidique, lequel procédé comprend :
    (a) l'agencement des membres du répertoire immunologique peptidique dans au moins deux séries de lignes continues dans lesquelles chaque ligne desdites première et deuxième séries comprend un membre dudit répertoire immunologique peptidique et de telle sorte que sensiblement tous les différents membres du répertoire immunologique peptidique soient juxtaposés les uns aux autres.
    (b) la détection de l'interaction/des interactions aux intersections des différents membres du répertoire immunologique peptidique.
  48. Procédé selon la revendication 46 ou 47, lequel procédé utilise le système double hybride de levure pour identifier les membres des répertoires immunologiques de molécules qui interagissent les uns avec les autres.
  49. Procédé de génération (et éventuellement de criblage) d'une bibliothèque de doubles hybrides de levure constituée sensiblement de tous les membres d'un premier répertoire immunologique de polypeptides appariés sensiblement à tous les membres d'un deuxième répertoire immunologique de polypeptides, lequel procédé comprend:
    (a) l'agencement de cellules de levure contenant une pluralité de séquences nucléotidiques pour générer au moins une matrice, dans lequel les séquences nucléotidiques sont agencées dans au moins une première série de lignes continues dans laquelle chaque ligne de ladite première série comprend des cellules de levure contenant une séquence nucléotidique et l'agencement du deuxième répertoire immunologique dans au moins une deuxième série de lignes continues dans laquelle chaque ligne de ladite deuxième série comprend des cellules de levure contenant un membre dudit deuxième répertoire immunologique, dans lequel les premier et deuxième répertoires immunologiques forment au moins une matrice, de telle sorte que sensiblement tous les membres du premier répertoire immunologique soient juxtaposés sensiblement à tous les membres du deuxième répertoire immunologique ;
    (b) le fait de permettre aux cellules de levure contenant les membres du premier répertoire immunologique de s'apparier aux cellules de levure contenant les membres du deuxième répertoire immunologique là où les deux répertoires immunologiques s'entrecroisent ; et éventuellement
    (c) l'expression des séquences nucléotidiques pour produire les polypeptides correspondants du premier et du deuxième répertoires immunologiques ; et éventuellement
    (d) la détection de l'interaction/des interactions entre les membres polypeptidiques du premier et du deuxième répertoires immunologiques.
EP01943634A 2000-06-23 2001-06-22 Procede de depistage par une matrice Expired - Lifetime EP1292710B1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
GB0015443 2000-06-23
GB0015443A GB0015443D0 (en) 2000-06-23 2000-06-23 Matrix screening method
GB0026099 2000-10-25
GB0026099A GB0026099D0 (en) 2000-10-25 2000-10-25 Matrix screening method
US24685100P 2000-11-08 2000-11-08
US246851P 2000-11-08
PCT/GB2001/002831 WO2001098534A2 (fr) 2000-06-23 2001-06-22 Procede de depistage par une matrice

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EP1292710B1 true EP1292710B1 (fr) 2007-08-29

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JP (1) JP2004501379A (fr)
AT (1) ATE371749T1 (fr)
AU (1) AU2001266172A1 (fr)
CA (1) CA2410857A1 (fr)
DE (1) DE60130211T2 (fr)
DK (1) DK1292710T3 (fr)
ES (1) ES2292596T3 (fr)
PT (1) PT1292710E (fr)
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US11466265B2 (en) 2020-05-11 2022-10-11 A-Alpha Bio, Inc. High-throughput screening methods to identify small molecule targets
US11474111B2 (en) 2020-06-01 2022-10-18 A-Alpha Bio, Inc. et al Methods for characterizing and engineering protein-protein interactions
US11820970B2 (en) 2016-01-15 2023-11-21 University Of Washington High throughput protein-protein interaction screening in yeast liquid culture

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EP1360490B1 (fr) 2001-01-23 2011-12-21 President and Fellows of Harvard College Reseaux de proteines a acide nucleique programmable
US8178316B2 (en) 2006-06-29 2012-05-15 President And Fellows Of Harvard College Evaluating proteins
US8716437B2 (en) 2007-12-07 2014-05-06 Steven A. Goldstein Identification of toxin ligands
KR20120122869A (ko) 2009-06-02 2012-11-07 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 신경변성 질환을 갖는 대상에서 항체에 의해 인식되는 소분자의 확인법
WO2011047257A1 (fr) 2009-10-16 2011-04-21 The Board Of Regents Of The University Of Texas System Compositions et procédés de production de banques de peptoïdes cycliques
BR112015017560A2 (pt) * 2013-01-24 2018-05-15 Scripps Korea Antibody Institute biblioteca de fv à base de combinação de proteína e modo de preparação da mesma
WO2024071112A1 (fr) * 2022-09-27 2024-04-04 国立大学法人浜松医科大学 Procédé de production d'anticorps

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DE60043937D1 (de) * 1999-06-26 2010-04-15 Odyssey Thera Inc Eine in vivo "bibliothek-gegen-bibliothek-selektion" von optimierten protein-protein-wechselwirkungen

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Publication number Priority date Publication date Assignee Title
US11820970B2 (en) 2016-01-15 2023-11-21 University Of Washington High throughput protein-protein interaction screening in yeast liquid culture
US11466265B2 (en) 2020-05-11 2022-10-11 A-Alpha Bio, Inc. High-throughput screening methods to identify small molecule targets
US11474111B2 (en) 2020-06-01 2022-10-18 A-Alpha Bio, Inc. et al Methods for characterizing and engineering protein-protein interactions
US11726097B2 (en) 2020-06-01 2023-08-15 A-Alpha Bio, Inc. Methods for characterizing and engineering protein-protein interactions

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EP1292710A2 (fr) 2003-03-19
DE60130211T2 (de) 2008-05-29
ES2292596T3 (es) 2008-03-16
DE60130211D1 (de) 2007-10-11
AU2001266172A1 (en) 2002-01-02
PT1292710E (pt) 2007-12-04
CA2410857A1 (fr) 2001-12-27
DK1292710T3 (da) 2007-12-27
WO2001098534A3 (fr) 2002-05-30
JP2004501379A (ja) 2004-01-15
WO2001098534A2 (fr) 2001-12-27
ATE371749T1 (de) 2007-09-15

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